1. Field of the Invention
The present invention relates to enzyme immunoassay techniques for the detection of bindable substances, such as antibodies and antigens, and particularly in home diagnostic kits. The invention further relates to the use of chromogenic substances in enzyme immunoassays.
2. Description of the Prior Art
There are a number of immunoassay techniques in contemporary use for laboratory detection and measurement of antigens or antibodies present within a test sample. Most of these techniques, however, are unsuitable for use outside a laboratory setting because of complexity of the detection equipment and other difficulties inherent in conducting many conventional immunoassay techniques. Thus, there is a need for a technique suited for home diagnostic immunoassay kits which may be readily used, for example, for the detection of antigens such as human choriogonadotropin hormone (HCG, immunogen) which is present in the urine of pregnant women. In order for a diagnostic immunoassay kit to be satisfactory for home use, the kit must be relatively inexpensive, the immunoassay method must be easy to use, reliable, and efficient, and above all must be safe. Additionally, the test method must be of sufficient sensitivity to easily detect the desired antigen in the test sample.
The earliest conventional immunoassay methods for detection and measurement of antigens or antibodies in a test sample are the radioimmunoassay method (RIA) and the fluorescent immunoassay technique (FIA). In the radioimmunoassay method, the antigen or antibody to be detected is either directly or indirectly labelled with radioactive isotope, commonly an isotope of iodine. Although a radioimmunoassay generally exhibits a high degree of sensitivity even for detection of trace amounts of test antigen or antibody, these tests all involve the use of hazardous radioactive materials which require special handling, storage, and disposal. Also, expensive analytical equipment is required, particularly in radioimmunoassay methods which involve the precipitation of immune complexes, which requires detailed analytical recovery techniques. Solid phase radioimmunoassays circumvent the need for detailed analytical recovery required in the precipitation method, but require much longer incubation times, typically between 30 to 60 hours. Therefore, in view of the potential hazard involved in handling radioactive material, and the need for expensive detection equipment and long incubation times, the radioimmunoassay method is unsuitable for application to home diagnostic kits.
In the immunofluorescence assay the test antigen or antibody may be labelled directly or indirectly by use of fluorescent dyes (fluorochromes) such as fluorscein and rhodamine which can be coupled to the test antigen or antibodies or their immunocomplexes without destroying their specificity. Such conjugates labelled with fluorescent dye can be visualized in a fluorescence microscope. Major disadvantages of the immunofluorescence method either by the direct or indirect method are firstly the dependence on expensive fluorescence microscopes for detecting the labelled conjugate, and secondly, the acknowledged difficulty in quantifying the test antibody or antigen present in the sample. Therefore, the immunofluorescence assay method is unsuitable for use in connection with home diagnostic kits.
In recent years, the enzyme immunoassay method has received increasing attention from researchers for use in detecting and measuring antibodies or antigen in test samples. The enzyme immunoassay methods involve enzyme labelling of the test antigen or antibody either directly or indirectly by labelling immunocomplexes which bind specifically to the test antigen or antibody and which catalyze reaction with a substrate. Some means is provided for monitoring enzyme activity. For example, in the measurement of the enzyme activity of oxidoreductases, one might monitor the oxidation of a chromogenic substance by a substrate such as hydrogen peroxide. Such so-called colorometric assays are readily adapted to the home-testing environment. When the chromogenic substance oxidizes, it forms a chromophore which exhibits visually discernable color changes.
Typical enzyme immunoassays include competitive EIA for antigens, and an enzyme linked immunosorbent assay (ELISA) which also includes direct and indirect ELISA methods. In the competitive EIA method, antigen labelled with enzyme competes with unlabelled sample antigen for binding to a limited quantity of antibodies which have been adsorbed onto a support medium. Once the amount of bound enzyme labelled antigen has been determined, the amount of sample antigen can be determined by the difference between the total amount of antibody bound to antigen less the amount of antibody bound to labelled antigen.
In enzyme immunoassay methods, antibodies specific to the test antigen may be first adsorbed in excess amount onto a solid surface such as a plastic well or tube. The test solution containing antigen is then added; the antigen will bind to the adsorbed antibody. The solid phase, that is the phase composed of all material bound to the antibody, is then thoroughly washed to separate unbound components. Further steps are directed toward quantifying the bound antigen. In the double sandwich antibody ELISA method, an enzyme labelled second antibody, preferably having binding sites different from those of the first antibody, is added and reacts with specific determinant sites on the bound antigen. The enzyme labelled second antibody is added in excess to assure that all the antigen present in the solid phase that is bound to the first antibody will also be bound to enzyme labelled second antibody. The enzyme labelled second antibody molecules will bind in a fixed ratio to each antigen molecule depending on the valence, i.e. specific available binding sites, of the antigen for the second antibody. The solid phase is again washed to remove excess second antibody and any other unbound constituents. An enzyme substrate is then added in solution in excess amount, whereby it makes contact with the bound solid phase. For the enzyme horseradish peroxidase, the substrate may typically be composed of a solution of hydrogen peroxide and a chromogenic material.
o-Phenylenediamine (OPD) heretofore has been acknowledged as one of the most sensitive chromogenic substrates available for detection of peroxidase activity. However, OPD produces a yellowish/orange chromophore which although discernible to the unaided eye is nonetheless not a preferred color for a chromophore, since the eye is more sensitive to other colors in the light spectrum such as blue. Other conventional chromogenic compounds having good sensitivity for peroxidase enzyme detection in enzyme linked immunoassays are o-tolidine and ABTS [2,2'-azinodi(3-ethylbenzothiazolinesulfone-6) diammonium salt]. Although o-tolidine and ABTS have been used successfully for detection of a number of specific antigens using ELISA methods, these latter two chromogenic compounds each have less sensitivity than OPD. All of these chromogenic compounds have been reported as soluble and initially colorless, yielding color change upon oxidation with hydrogen peroxide. Typical enzymes that have been used in the enzyme immunoassay methods are horseradish peroxidase, glucose oxidase, .beta.-D-galactosidase, and alkaline phosphatase. However, since the latter two are found in normal human urine, they are not preferred for use in connection with enzyme immunoassay techniques if they are to be applied in home diagnostic kits. The amount of test antigen present in the solid phase of the double sandwich ELISA method is then directly measurable after the chromogenic substrate has been added, since when there is excess substrate the rate of color change of the chromogen is independent of the substrate concentration and is a function of the total enzyme concentration. The enzyme concentration is a function of the amount of enzyme labelled second antibody, which in turn is a function of the amount of test antigen. Therefore, the rate of color change is a function of the amount of test antigen. The rate of color change can be measured by means of a spectrophotometer if quantification of the amount of test antigen is desired. For use in home diagnostic kits when quantification is not required, the assay should be capable of permitting the user to detect a color change visually which in turn would indicate the presence of a specific antigen in the test sample.
Although the above-described enzyme immunoassay method has been conventionally used in recent years in assaying for antigen or antibodies in a laboratory setting, there has been some difficulty in applying these methods to home diagnostic kits, e.g. for the detection of gonococcus (GC), human chorionic gonadotropin (HCG), or human luteinizing hormone (HLH). Conventional chromogenic reagents preferably are prepared in fresh batches just prior to use, and tend to oxidize and become colored spontaneously when left in storage, typically even for as little as one hour.
In general, a chromogenic compound for detection of enzymes such as horseradish peroxidase should be relatively inexpensive, easy to use in connection with home diagnostic assays, and above all, noncarcinogenic and safe. The chromogenic compound importantly should be stable, soluble, and exhibit rapid color change upon reaction. Also, with substrate, e.g., hydrogen peroxide when exposed to oxidative enzymes, the product chromophore should likewise be safe, stable, and exhibit a high molar absorptivity.
Other chromogenic compounds have been used in pathological studies or assays outside the realm of enzyme immunoassay methods. For example, benzidine has been used to determine peroxidase activity of heme proteins. In such an application, benzidine-hydrogen peroxide chromogenic substrates have been used in forensic medicine for the detection of blood using the peroxidase activity of hemoglobin. Also, benzidine staining procedures have been used to detect the peroxidase activity of the heme proteins cytochrome P-450 and cytochrome P-420. Specifically, the peroxidase activity of these cytochromes has been detected on sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis by a benzidine staining procedure. Problems have been encountered, however, with the use of benzidine, one of the more important being that it has been found to be a potent human bladder carcinogen. Additionally, staining with benzidine may lack sensitivity. The stain exhibits limited stability, therefore making it difficult to photograph. Researchers in the field of forensic medicine have, therefore, sought alternatives to benzidine for the detection of peroxidase activity of heme proteins, in particular for detection of peroxidase activity of hemoglobin. One such alternative reported in the literature is the use of 3,3',5,5'-tetramethylbenzidine in hydrogen peroxide as a stain for the peroxidase activity of heme proteins, particulary cytochrome P-450. The results of the improved staining procedures using tetramethylbenzidine are reported in P. Thomas, B. Ryan, and W. Levin, Analytical Biochemistry 75, 168-176 (1976).
The advantages of using tetramethylbenzidine for the heme staining of cytochrome P-450 as reported in this reference were that the TMB substrates exhibited increased sensitivity, clear dull background, thereby improving color contrast, and greater staining stability, i.e., the TMB stained gels could be stored in the dark at room temperature for at least one month with only minimal loss in TMB stain intensity. In Thomas et al. supra., the improvement in stability of the TMB-hydrogen peroxide staining was reported to be in marked contrast to that obtained with benzidine-hydrogen peroxide where much of the stain is lost within one hour after heme staining for detection of cytochrome P-450. The TMB stained gels were reported to give distinct color even after 25 hours, in contrast to the results obtained with benzidine heme staining wherein much of the color was lost in only one hour after staining.
In the reference Thomas et al. supra., the preferred preparation of the TMB chromogenic solution for detecting the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate (SDS)-polyacrylamide gel was described as follows: A 6.3mM TMB solution was freshly prepared in methanol. Immediately before use, 3 parts of the TMB solution were mixed with 7 parts of 0.25 M sodium acetate buffer (pH 5.0). After 1 or 2 hours with occasional mixing (every 10-15 min.), H.sub.2 O.sub.2 was added to a final concentration of 30mM. The staining was visible within 3 minutes when using this solution. After the gels were stained, they were placed in an acetate buffered 30 percent isopropanol solution (i.e. the gels were placed in isopropanol: 0.25 M sodium acetate, pH 5.0 at a ratio of 3:7). This served to clear the gel background and enhance staining intensity and permitted storage of the stained gels in the dark at room temperature for at least 2 months with minimal loss in stain intensity.
The use of alternative solvents for TMB such as ethanol or isopropanol instead of methanol reportedly resulted in diminished stain intensity. The 3:7 ratio of methanol to sodium acetate buffer of pH 5.0 was found to be optimal. When the pH 5.0 sodium acetate buffer was replaced with buffers at pH 4.0, 4.5, 5.5 or 6.0, the stability and sensitivity of staining were significantly reduced.
A salt of tetramethylbenzidine, believed to be noncarcinogenic, namely tetramethylbenzidine dihydrochloride (TMB-d) has been reported in the literature and used as a suitable substitute for benzidine for staining of hemoglobin containing cells and for quantitative determination of hemoglobin in solutions. See, H. H. Liem, et al. Analytical Biochemistry, 98, 388-393 (1979). Unlike tetramethylbenzidine, TMB-d is water soluble obviating use of an organic solvent. It also dissolves in 10 percent acetic acid forming a green oxidation product. It has the disadvantage, however, of being unstable in the presence of moisture and air, thereby diminishing its effectiveness.
The use of tetramethylbenzidine-hydrogen peroxide chromogenic substrates as stains for detection of peroxidase activity of heme proteins, such as cytochromes, as reported in Thomas et al., or hemoglobin as reported in Liem is directed to the forensic sciences for detection of blood particularly in samples produced during criminal or accident investigations. The uses of tetramethylbenzidine disclosed in these references, including heme staining and staining of cytochrome or hemoglobulin for application to the forensic sciences, do not extend to the use as a chromogen in enzyme immunoassays.
The use of avidin-biotin complexes (ABC) linked to enzymes such as horseradish peroxidase are disclosed in the literature as increasing the sensitivity of enzyme immunoassays. This technique employs a primary antibody to which is bound test antigen. A biotinylated second antibody then reacts with the bound antigen. An avidin-biotin peroxidase complex (ABC) is prepared and reacts with the biotinylated secondary antibody. The peroxidase will catalyze reaction between chromogen and a substrate such as hydrogen peroxide to produce a color change revealing the presence of the test antigen. This application of the avidin-biotin complex has been used to assay for polypeptide hormones as reported by S. M. Hsu, L. Raine, and H. Fanger, "A Comparative Study of the Peroxidase-Antiperoxidase Method and An Avidin-Biotin Complex Method for Studying Polypeptide Hormones with Radioimmunoassay Antibodies", Am. J. Clinical Pathology 7: 734-738 (1981) and in S. M. Hsu and H. J. Ree, "SelfSandwich Method--An Improved Immunoperoxidase Technic for the Detection of Small Amounts of Antigen", Am. J. Clinical Pathology 74: 32-40 (1980). Thus, the ABC technique described in Hsu et al. results in a double antibody sandwich with antigen linked between the antibodies and wherein the second antibody is biotinylated; the second antibody in turn is linked to the avidin-biotin peroxidase.
Since the avidin molecule has four binding sites, there are potentially a greater number of enzyme molecules in the complex for each antigen molecule than if enzyme is linked directly to the second antibody. There may be, therefore, a greater sensitivity of this complex to chromogenic substrates since the greater number of enzyme molecules results in increased rate of color change, i.e., increased rate of reaction of chromogen to form color detectable chromophore.
Accordingly, it is an object of the present invention to provide a very sensitive, reliable immunoassay method suitable for use in a home diagnostic kit for detection of antigens.
Another object of the present invention is to provide an improved chromogenic substrate for use in immunoassay techniques for application to home diagnostic kits. A related object of the invention is to provide a safe chromogenic substrate which exhibits increased sensitivity to peroxidase activity, and does not lose its activity prior to use.